The instruction of Ni Sepharose
Purification Procedure—Native Conditions
The method described following is designed for purification of 6xHis-tagged recombinant
soluble proteins expressed in bacteria. The method could be referred if the target proteins
from other sources have a 6xHis structure in the end.
Packing of the Ni2+ affinity column
1．Gently invert the Ni2+ gel-type resins and take 2ml resins into the column. Sink the
resins naturally;
2．Wash the resins with 3 bed volumes sterile water (it remains only 1 ml of the 2ml
resins after sinking naturally).
3. Wash and equilibrate the resins with 3 bed volumes binding buffer (pH7.8). Place the
column on the table for purification of the His-tagged proteins.
4．Drain the binding buffer in the resins to the top of the column.
5．Add the supernatant obtained in the step 8 into the column. Adjust the flow rate to 10
bed volumes per hour (15-20ml).
6．Wash the resins with 6 bed volumes binding buffer（pH7.8）.
7．Wash the resins with 4 bed volumes binding buffer（pH7.8） until A280<0.01.
8. Elute the binding proteins by washing with 6 bed volumes elution buffer of 10mmol/L
imidazole. Collect the elutriant 1ml a step and monitor the value of A280.
9. Repeat the step 8 with elution buffer contained higher concentration of imidazole.
10. Analyze the protein components by SDS-PAGE with 10-20ul aliquot of proteins.
Reagents:
1．Binding buffer:（pH7.8）
20mmol/L phosphate buffer
500mmol/L NaCl
2．Elution buffer:（pH7.8）
20mmol/L phosphate buffer
500mmol/L NaCl
10mmol/L imidazole
3．Imidazole elution buffer:（pH7.8）
20mmol/L phosphate buffer
500mmol/L NaCl
Imidazole could be added into the buffers above to get imidazole elution buffer with
different imidazole concentration.
Purification Procedure—Denaturing Conditions
Inclusion bodies would be form if the proteins express level are very high or have a poor
solubility in the bacteria or other cells. Usually, it needs to use guanidine hydrochloride or
urea to dissolve the inclusion bodies. NTA has the advantage of binding the 6His-tagged
proteins in 6M guanidine hydrochloride compared to the MBP and GST. The whole
procedure can be done in the condition of guanidine hydrochloride. The purified proteins
should be refolded correctly to get the biological activity. The following method is designed
to purify the His-tagged proteins in inclusion bodies.
Packing of the Ni2+ affinity column
1．Gently invert the Ni2+ gel-type resins and take 2ml resins into the column. Sink the
resins naturally;
2．Wash the resins with 3 bed volumes sterile water (it remains only 1 ml of the 2ml
resins after sinking natural).
3. Wash and equilibrate the resins with 3 bed volumes binding buffer (pH7.8). Place the
column on the table for purification of the His-tagged proteins.
4．Drain the binding buffer in the resins to the top of the column.
5．Add the supernatant obtained in the step 3 into the column. Adjust the flow rate to 10
bed volumes per hour (15-20ml). Collect the elutriant for analyzing the protein
components by SDS-PAGE.
6．Wash the column with 6 bed volumes NTA-O Buffer (pH7.8).
7．Wash the column with 6 bed volumes NTA-10Buffer (pH7.8) until A280<0.01.
8. Elute the binding proteins by washing with 5 bed volumes elution buffer contained
different imidazole concentration. Collect the elutriant 1ml a step and monitor the value
of A280.
9．Make certain of the distribution of the target protein, the most effective method is
SDS-PAGE analyzing.
10. Make further purification of the target protein according to the purpose.
11．Select a suitable procedure that can refer to the programs in relation to refold the
purified target proteins.
Reagents:
NTA-0Buffer:
20mM Tris-HCl pH7.8，0.5M NaCl，10%Glycerol，6M guanidine hydrochloride
NTA-10Buffer:
20mM Tris-HCl pH7.8，0.5M NaCl，10%Glycerol，6M guanidine hydrochloride 10mM
Imidazole
20mM Tris-HCl。PH7.8，0.5M NaCl，10%Glycerol，6M guanidine hydrochloride。HCl
100mM Imidazole
NTA-XBuffer:
20mM Tris-HCl PH7.8，0.5M NaCl，10%Glycerol，6M guanidine hydrochloride ，XmM
Imidazole
Regeneration of the Ni-NTA Sepharose4B:
The binding efficiency of the Ni-NTA Sepharose4B will decline after several (3-5times)
purification cycles. The Sepharose could be regenerated in the following procedure to
extend the useful time and the binding efficiency of the Sepharose. The NTA Sepharose
almost could be used unlimitedly after a full clean-up and regeneration. Unless the
Sepharose is oxygenated or exhausted (the light blue disappear or the color turns to
yellow or brown).
The regeneration procedure of the NAT
1．Wash the Sepharose one time with 2 bed volumes stripping solution1 after the column
has been drained from the bottom of the column.
2．Wash the Sepharose with 5 bed volumes of deionized water;
3. Wash the Sepharose with 2 bed volumes stripping Soluting 2;
4．Wash the Sepharose with 1 bed volumes 25% ethanol;
5. Wash the Sepharose with 1 bed volumes 50% ethanol;
6. Wash the Sepharose with 1 bed volumes 75% ethanol;
7. Wash the Sepharose with 5 bed volumes100% ethanol;
8. Wash the Sepharose with 1 bed volumes 75% ethanol;
9. Wash the Sepharose with 1 bed volumes 50% ethanol;
10. Wash the Sepharose with 1 bed volumes 25% ethanol;
11. Wash the Sepharose with 1 bed volumes deionized water;
12. Wash the Sepharose with 5 bed volumes stripping Solution 3;
13. Wash the Sepharose with 1 bed volumes deionized water;
14. Wash with less than 2 bed volumes Ni Charging Solution, wash with water, and then
equilibrate the Sepharose with appropriate amount of the balance buffer (the color of the
Sepharose should be white after washing with EDTA or eluting the proteins with EDTA
and the color will be light blue after regeneration);
15．It should be stored in 20% ethanol at 4 ℃ for long term storage. Process the 14 steps
above before purification another time.
Reagents:
Stripping Solution 1：0.2M acetic acid/6M guanidine hydrochloride
Stripping Solution 2：2%SDS
Stripping Solution 3：0.1M EDTA pH8.0
Ni Charging Solution：100mM NiSO4